Freeze-Dried Product for Introducing Nucleic Acid, Oligonucleic Acid or Derivative Thereof

ABSTRACT

A freeze-dried product is provided which, together with demonstrating satisfactory ability to express function when used to introduce a gene or antisense nucleic acid and the like, enables concentration to be adjusted easily, offers easy handling and has superior storage performance. 
     A freeze-dried product of a complex containing a nucleic acid, oligonucleic acid or derivative thereof; a cationic polymer, or cationic lipid or aggregate containing the same; and, an anionic polymer; a method for preparing the freeze-dried product, comprising a step of forming a complex by mixing a nucleic acid, oligonucleic acid or derivative thereof; a cationic polymer, or cationic lipid or aggregate containing the same; and an anionic polymer, and a step of freeze-drying the complex; a preparation, reagent or kit containing the freeze-dried product for introducing a nucleic acid, oligonucleic acid or derivative thereof; and, a method for introducing a nucleic acid, oligonucleic acid or derivative thereof into cells that uses the freeze-dried product.

TECHNICAL FIELD

The present invention relates to a freeze-dried product of a complexcontaining a nucleic acid, oligonucleic acid or derivative thereof; acationic polymer, or cationic lipid or aggregate containing the same;and, an anionic polymer, for the purpose of introducing a nucleic acid,oligonucleic acid or derivative thereof into cells, a preparation methodof the same, and a preparation, reagent and kit for introducing anucleic acid, oligonucleic acid or derivative thereof containing thesame.

BACKGROUND ART

Gene therapy and antisense treatment methods are currently usedpractically for treating congenital genetic diseases, cancer cells orAIDS by introducing an intended gene, antisense oligonucleic acid orderivative thereof into cells and expressing that gene or function, andstudies are being conducted on various types of vectors for use ascarriers for introducing genes (DNA), antisense oligonucleic acids andderivatives thereof into cells.

Research is being conducted on cationic substances such as cationicpolymers, cationic liposomes and cationic lipids for use as one suchtype of vector in the form of a non-viral vector that eliminatesconcerns over safety, has favorable efficiency, is free ofimmunogenicity and is easily prepared.

In methods using these cationic substances, since complexes of DNA andcationic substances are positively charged, aggregation ends upoccurring due to interaction with blood cells and blood components suchas albumin, thereby impairing the delivery to the target cells. Studieshave been conducted on various methods to solve this problem, includingcoating a complex of nucleic acid and cationic polymer with a hyaluronicacid derivative (Patent Document 1: Japanese Unexamined PatentPublication No. 2005-176830); coating with polyethylene glycol (PEG)having a carboxyl group-containing side chain and sugarresidue-containing side chain (Patent Document 2: Japanese UnexaminedPatent Publication No. 2003-231748); and preventing complex aggregationby using PEG having a free carboxylic acid pendant group (Non-PatentDocument 1: J. Biomater. Sci. Polymer Edn., Vol. 14, No. 6, pp. 515-531(2003)).

Complexes of DNA and cationic substances modified in this manner exhibitlow aggregation and exhibit favorable gene expression in cells. However,since these complexes are heterogeneous suspensions, they have poorstorage performance, are required to be used promptly followingpreparation, and end up aggregating when prepared at highconcentrations, thereby having the disadvantages of difficulty inadjusting concentration and bothersome handling. In addition, it wasdifficult to prepare these complexes with satisfactory reproducibility.

On the other hand, in the case of using the modified complex of DNA andcationic substance as described above to introduce a gene and the like,it is also important to control the size (particle diameter) of thecomplex. This is because, in the case of administering into blood ortissue, subsequent diffusion of the complex and the efficiency at whichit is delivered and incorporated by cells greatly affect itspharmacological efficacy. In general, however, in the case of mixingionic polymers such as cationic polymers to form a complex, the polymerends up aggregating resulting in an increased likelihood of theformation of extremely large particles or fibrous complexes. In order toprevent this, it is necessary to make the concentrations of thesolutions mixed extremely dilute. However, since preparations used forgene introduction and the like are required to have a certain minimalconcentration, there was the problem of being unable to avoid theformation of large mass of aggregates. In addition, although methods arealso considered consisting of first forming small complexes by mixingdilute solutions followed by concentrating, suitable means forconcentration was unable to be achieved since the complex particlesended up rapidly aggregating.

Therefore, in order to solve these problems, studies were conducted onfreeze-drying methods typically used to facilitate transport ofbiological preparations and enhance storage stability. However, sincefreeze-drying complexes of nucleic acids, oligonucleic acids orderivatives thereof with polycationic substances ends up impairment ofthe structure of the complex due to the freeze-drying, when used forgene introduction or introduction of oligonucleic acids, the complexeswere confirmed to hardly demonstrate any functions as genes or antisenseoligonucleic acids (Non-Patent Document 2: J. Pharm. Sci., Vol. 90, pp.1445-1455 (2001)).

A method to solve these problems was proposed in which freeze-drying iscarried out after adding a high concentration of monosaccharide ordisaccharide (Non-Patent Document 3: Biochim. Biophys. Acta., 2000 Sep.29, 1468 (1-2): 127-138). However, the amount of sugar required is 500to 1000 times the amount of DNA in terms of the weight ratio, makingthis method impractical in consideration of the solution followingrehydration having a much higher osmotic pressure than physiologicalconditions. In addition, monosaccharides and disaccharides do not offeradvantageous effects for gene expression. In addition, the use of aneutral polysaccharide, dextran has been attempted to reduce osmoticpressure after rehydration (Non-Patent Document 4: J. Pharm. Sci., Vol.94, pp. 1226-1236 (2005)). However, high molecular weight dextrangreatly inhibits gene expression, and in the case of using low molecularweight dextran (molecular weight of about 3000), it was necessary to adddextran at a considerably high concentration of 100 times or more thatof DNA in terms of the weigh ratio in order to prevent aggregationcaused by freeze-drying (Non-Patent Document 4: J. Pharm. Sci., Vol. 94,pp. 1226-1236 (2005)). For use of this type of freeze-dried product invivo, the freeze-dried product is required to be rehydrated with a smallamount of water or solvent after freeze-drying to obtain the requiredconcentration of DNA and then concentrated to a high concentration. As aresult, the concentration of dextran following rehydration exceeds 10%,and there are limitations during the freeze-drying procedure such as onDNA concentration and cooling temperature, thereby making practicalapplication difficult.

Patent Document 1: Japanese Unexamined Patent Publication No.2005-176830

Patent Document 2: Japanese Unexamined Patent Publication No.2003-231748

Non-Patent Document 1: J. Biomater. Sci. Polymer Edn., Vol. 14, No. 6,pp. 515-531 (2003)

Non-Patent Document 2: J. Pharm. Sci., Vol. 90, pp. 1445-1455 (2001)

Non-Patent Document 3: Biochim. Biophys. Acta., 2000 Sep. 29, 1468(1-2): 127-138

Non-Patent Document 4: J. Pharm. Sci., Vol. 94, pp. 1226-1236 (2005)

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

The inventors of the present invention conducted extensive studies toovercome the aforementioned problems, and as a result they found that ifa freeze-dried product of a complex containing a nucleic acid,oligonucleic acid or derivative thereof; a cationic polymer, or cationiclipid or aggregate containing the same; and, an anionic polymer isintroduced into cells, the introduced gene, oligonucleic acid orderivative thereof satisfactorily expresses the function thereof,thereby leading to completion of the present invention.

Means for Solving the Problems

The present invention relates to a freeze-dried product of a complexcontaining a nucleic acid, oligonucleic acid or derivative thereof; acationic polymer, or cationic lipid or aggregate containing the same;and, an anionic polymer. In addition, the present invention relates to apreparation, reagent and kit containing the freeze-dried product forintroducing a nucleic acid, oligonucleic acid or derivative thereof.Moreover, the present invention relates to a method for preparing thefreeze-dried product comprising a step of forming a complex by mixing anucleic acid, oligonucleic acid or derivative thereof; a cationicpolymer, or cationic lipid or aggregate containing the same; and ananionic polymer, followed by a step of freeze-drying the complex. Thepresent invention also relates to a method for introducing a gene,oligonucleic acid or derivative thereof into cells, the method using thefreeze-dried product.

EFFECTS OF THE INVENTION

The freeze-dried product of the present invention enables concentrationto be adjusted easily, offers easy handling and has superior storageperformance. In addition, since the freeze-dried product of the presentinvention contains an anionic polymer, a stable dispersion containing acomplex of an extremely small size can be obtained at an arbitraryconcentration even when rehydrating with a solvent to form a suspensionor dilution at the time of use. Moreover, a nucleic acid, oligonucleicacid or derivative thereof can be efficiently introduced into cellswithout causing aggregation even during gene introduction, andsatisfactory ability to express a function thereof is demonstrated byvarious types of administration methods such as local administration orintravenous administration.

BEST MEANS FOR CARRYING OUT THE INVENTION

The freeze-dried product of the present invention is a freeze-driedproduct of a complex containing a nucleic acid, an oligonucleic acid ora derivative thereof; a cationic polymer, or cationic lipid or aggregatecontaining the same; and an anionic polymer. A nucleic acid,oligonucleic acid or derivative thereof in the complex forms a complexby ionic bonding with a cationic polymer, or cationic lipid or aggregatecontaining the same, and the cationic polymer or cationic lipid arefurther ionicly bonded with an anionic polymer. These components form acomplex that is mainly coated with anionic polymer depending on themixing ratio, mixing sequence and the like.

Any nucleic acid or oligonucleic acid and the like introduced for thepurpose of gene therapy or antisense therapy can be used for the nucleicacid, oligonucleic acid or derivative thereof able to be used in thefreeze-dried product of the present invention, specific examples ofwhich include various types of nucleic acids, oligonucleic acids andderivatives thereof, such as various DNA and RNA (single-strand ordouble-strand) (such as plasmid DNA, double-stranded oligo RNA, mRNA,tRNA, rRNA or cDNA), sense or antisense oligonucleotides (includingrecombinants) and derivatives thereof, or ribozymes or mixtures thereof.In addition, the base portion or sugar portion of these nucleic acidsmay be modified or substituted as necessary. For example, plasmid DNAcan be used preferably in the case of a nucleic acid, and oligo DNA or aderivative thereof in the form of S-oligo, double-stranded RNA for RNAinterference or ribozyme RNA can be used preferably in the case of anantisense nucleic acid. Among them, plasmid DNA can be used particularlypreferably.

Positively charged, naturally-occurring or synthetic polymers having amolecular weight of about 1,000 to 3,000,000 and having a plurality of,preferably five or more, functional groups capable of forming a complexwith DNA in water can be used for the cationic polymer able to be usedin the freeze-dried product of the present invention, and examples ofsuch functional groups include organic amino groups such as optionallysubstituted amino groups, ammonium groups or salts thereof (and thesegroups may be mono- or poly-substituted with, for example, alkyl groupshaving 1 to 6 carbon atoms, phenyl groups and the like), imino groups,imidazolyl groups or guanidino groups. Examples of such cationicpolymers include positively charged proteins and polypeptides;positively charged dendrimers; positively charged synthetic polymers;and positively charged polysaccharide derivatives or salts thereof andcombinations thereof.

The molecular weight of positively charged proteins or positivelycharged polypeptides able to be used as cationic polymers in thefreeze-dried product of the present invention is preferably about 1,000to 500,000. Specific examples of such proteins and polypeptides includeproteins or polypeptides such as protamine, histone, HelΔ1 or gelatin.In addition, examples also include polyamino acids containing positivelycharged amino acid residues. Specific examples of such polyamino acidscontaining positively charged amino acid residues include poly-L-lysine,polyarginine and polyornithine. Examples of salts of these proteins andpolypeptides include hydrochlorides, sulfates, phosphates and borates.

Positively charged dendrimers having functional groups like thosedescribed above able to be used as cationic polymers refer to dendrimershaving an optionally substituted amino group, ammonium group or saltthereof (and these groups may also be mono- or poly-substituted with,for example, alkyl groups having 1 to 6 carbon atoms, phenyl groups andthe like) on the terminal or within a branched molecular chain, and themolecular weight thereof is preferably about 1,000 to 500,000. Specificexamples of dendrimers include polyamide amine dendrimers and polylysinedendrimers. In addition, examples of dendrimer salts includehydrochlorides, sulfates, phosphates and borates.

Positively charged synthetic polymers able to be used as cationicpolymers are synthetic polymers having a plurality of, preferably fiveor more, functional groups capable of forming a complex with DNA inwater in a molecule thereof as previously described, and preferablyhaving a molecular weight of 1,000 to 3,000,000. Specific examples ofsynthetic polymers include polyethyleneimines (including linearpolyethyleneimines and branched polyethyleneimines), polymers orcopolymers of 2-dimethylaminoethyl methacrylate, and polymers orcopolymers of 2-trimethylaminoethyl methacrylate. The molecular weightof one example of synthetic polymers in the form of polyethyleneiminesis preferably about 1,000 to 500,000, more preferably about 5,000 to200,000 and most preferably about 10,000 to 100,000. In addition,examples of salts of polyethyleneimines include hydrochlorides,sulfates, phosphates and borates.

Positively charged polysaccharide derivatives able to be used ascationic polymers are polysaccharide derivatives having a plurality of,preferably five or more, functional groups capable of forming a complexwith DNA in water in a molecule thereof, and having a molecular weightof preferably 1,000 to 3,000,000 and more preferably 5,000 to 500,000.Specific examples of such polysaccharides include chitosan and dextranderivatives introduced with functional groups as previously described.Among these, the molecular weight of chitosan is preferably about 1,000to 500,000, more preferably about 5,000 to 200,000 and most preferablyabout 10,000 to 100,000. Examples of salts of chitosan includehydrochlorides and acetates. In addition, the molecular weight ofdextran derivatives is preferably 3,000 to 1,000,000. Specific examplesof such dextran derivatives include diethylaminoethyl dextran.

Even though the aforementioned cationic polymers are originally notpositively charged, they can be used provided they become positivelycharged as a result of introducing a functional group such as an aminogroup, and may also be further modified with a sugar chain, oligopeptideor antibody and the like as necessary.

Examples of cationic lipids (including cationic cholesterol derivatives)able to be used in the freeze-dried product of the present inventioninclude DC-Chol (3β-(N—(N′,N-dimethylaminoethane)carbamoyl)cholesterol),DDAB (N,N-distearyl-N,N-dimethylammonium bromide), DMRI(N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide), DODAC (N,N-dioleyl-N,N-dimethylammonium chloride), DOGS(diheptadecylamidoglycylspermidine), DOSPA(N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoroacetate), DOTAP(N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride) andDOTMA (N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride) orcombinations thereof.

In addition, mixtures of the aforementioned cationic lipids (such asDOSPA) and neutral substances such as DOPE(dioleylphosphatidylethanolamine) or cholesterol can be used asaggregates containing cationic lipids. Preferable examples of aggregatescontaining cationic lipids include lipofectamine (liposome containing3:1 w/w mixture of DOSPA and DOPE), lipofectin (liposome containing 1:1w/w mixture of DOTMA and DOPE) and mixtures thereof.

In the freeze-dried product of the present invention,polyethyleneimines; protamine; HelΔ1; dendrimers such as polyamide aminedendrimers or polylysine dendrimers; chitosan; polymers or copolymers of2-dimethylaminoethyl methacrylate or polymers or copolymers of2-trimethylaminoethyl methacrylate can be used preferably as cationicpolymer, while polyethyleneimines, polyamide amine dendrimers,polylysine dendrimers or chitosan can be used particularly preferably.In addition, lipofectamine (liposome containing a 3:1 w/w mixture ofDOSPA and DOPE) can be used preferably as a cationic lipid or aggregatecontaining the same.

Negatively charged, naturally-occurring or synthetic polymers containingan anionic group in a molecule thereof, having a molecular weight ofabout 500 to 4,000,000, and having a plurality of, preferably five ormore, functional groups in a molecule thereof capable of forming acomplex with a polycation in water can be used for the anionic polymerused in the freeze-dried product of the present invention, and examplesof such functional groups include a carboxyl group, —OSO₃H group, —SO₃Hgroup, phosphate group and salts thereof. Examples of such anionicpolymers include amphoteric polymers.

Polysaccharides and derivatives thereof having functional groupsselected from a carboxyl group, —OSO₃H group, —SO₃H group, phosphategroup and salts thereof; polyamino acids containing an amino acidresidue having negatively charged side chains; PEG derivatives havingcarboxyl side chains; synthetic polymers having functional groupsselected from the group consisting of a carboxyl group, —OSO₃H group,—SO₃H group, phosphate group and salts thereof; polymers havingfunctional groups selected from a carboxyl group, —OSO₃H group, —SO₃Hgroup, phosphate group and salts thereof, as well as optionallysubstituted amino groups, ammonium groups or salts thereof (and thesegroups may be mono- or poly-substituted with, for example, alkyl groupshaving 1 to 6 carbon atoms, phenyl groups and the like); andcombinations thereof can be used as an anionic polymer in thefreeze-dried product of the present invention.

Glucosaminoglycans can be preferably used as a polysaccharide orderivative thereof having functional groups as described above able tobe used as an anionic polymer in the freeze-dried product of the presentinvention. The molecular weight of such glucosaminoglycans is preferably1,000 to 4,000,000 and more preferably 4,000 to 3,000,000. Specificexamples of such glucosaminoglycans include hyaluronic acid,chondroitin, chondroitin sulfate, keratan sulfate, heparin and dermatansulfate. Among them, hyaluronic acid can be used particularlypreferably. Hyaluronic acid can also be used in the form of a salt ornegatively charged derivative thereof. Although the molecular weightthereof may be 5,000 or more, it is preferably 10,000 or more and morepreferably 100,000 to 3,000,000. Examples of salts of hyaluronic acidinclude sodium salts, potassium salts and ammonium salts. In addition,examples of derivatives of hyaluronic acid include those obtained byintroducing polyethylene glycol, peptide, sugar, protein, hydroiodicacid, antibody or portions thereof into hyaluronic acid, and amphotericderivatives having a positively charged portion by introducing spermine,spermidine, and the like are also included.

Polyamino acids containing an amino acid residue having a negativelycharged side chain able to be used as an anionic polymer in thefreeze-dried product of the present invention are polyamino acidspreferably having a molecular weight of 500 to 1,000,000 and containingan amino acid residue having as a side chain thereof a carboxyl group,—O—SO₃H group, —SO₃H group, phosphate group or salt thereof. Specificexamples of such polyamino acids include polyglutamic acid andpolyaspartic acid.

PEG derivatives having a carboxyl side chain able to be used as ananionic polymer in the freeze-dried product of the present invention arePEG derivatives having a molecular weight of 500 or more, preferably2,000 or more and more preferably 4,000 to 40,000 and having a pluralityof, preferably five or more, carboxyl side chains per molecule of PEG.PEG derivatives having carboxyl side chains can also be used as saltsthereof or negatively charged derivatives thereof. Examples of thesesalts include sodium salts, potassium salts and ammonium salts. Specificexamples of such PEG derivatives include the PEG derivatives describedin Non-Patent Document 1 (J. Biomater. Sci. Polymer Edn. Vol. 14, pp.515-531 (2003)).

Synthetic polymers having functional groups selected from a carboxylgroup, —O—SO₃H group, —SO₃H group, phosphate group and salts thereofable to be used as an anionic polymer in the freeze-dried product of thepresent invention are polymers or copolymers having a plurality of,preferably five or more, functional groups selected from a carboxylgroup, —O—SO₃H group, —SO₃H group, phosphate group and salts thereof permolecule thereof, and preferably having a molecular weight of 500 to4,000,000. Specific examples of such polymers or copolymers includepolymers or copolymers of acrylic acid or methacrylic acid having amolecular weight of 1000 to 3,000,000, sulfuric acid esters of polyvinylalcohol, and succinylated poly-L-lysine.

Polymers having functional groups selected from a carboxyl group,—O—SO₃H group, —SO₃H group, phosphate group and salts thereof, as wellas optionally substituted amino groups, ammonium groups or salts thereof(and these groups may be mono- or poly-substituted with alkyl groupshaving 1 to 6 carbon atoms, phenyl groups and the like), able to be usedas an anionic polymer in the freeze-dried product of the presentinvention are polymers having a molecular weight of 500 or more,preferably 2,000 or more and more preferably 4,000 to 40,000 and havinga plurality of, preferably five or more, functional groups selected froma carboxyl group, —OSO₃H group, —SO₃H group, phosphate group and saltsthereof per molecule thereof, as well as optionally substituted aminogroups, ammonium groups or salts thereof as previously described.Preferable examples of such polymers include PEG derivatives havingcarboxyl group side chains and an equivalent amount or less of theaforementioned amino groups, ammonium groups or salts thereof, andspecific examples include PEG derivatives able to be prepared using themethod described in Non-Patent Document 5 (Macromol. Biosci., Vol. 2,pp. 251-256 (2002)).

Anionic polymers able to be used in the freeze-dried product of thepresent invention can be used even if they are usually not negativelycharged provided they are made to be negatively charged by introducingfunctional groups such as a carboxyl group, and may be further modifiedwith a sugar chain, oligopeptide or antibody and the like as necessary.

In the freeze-dried product of the present invention, hyaluronic acid,PEG derivatives having carboxyl side chains, anionic polymers such aspolyacrylic acid or salts thereof can be preferably used as anionicpolymers, while hyaluronic acid, PEG derivatives having carboxyl sidechains or salts thereof can be used particularly preferably.

In addition, the use of an anionic polymer having the ability tospecifically adhere to target cells for introducing a nucleic acid makesit possible to specifically introduce a nucleic acid into the targetcells. For example, in the case of using hyaluronic acid for the anionicpolymer, cells having cell surface molecules such as CD44, whichspecifically bond with hyaluronic acid, can be targeted. In addition,the use of an anionic polymer introduced with RGD peptide makes itpossible to target numerous types of tumor cells, while the use of ananionic polymer introduced with a galactose side chain makes it possibleto target liver cells or cells originating in the liver.

In the freeze-dried product of the present invention, preferableexamples of combinations of a cationic polymer or cationic lipid oraggregate containing the same and an anionic polymer includepolyethyleneimines and hyaluronic acid; polyethyleneimines and PEGderivatives having carboxyl side chains; aggregates containing DOSPA(such as lipofectamine (liposome containing 3:1 w/w mixture of DOSPA andDOPE)) and hyaluronic acid; and aggregates containing DOSPA (such aslipofectamine) and PEG derivatives having carboxyl side chains.

Although varying according to the types of target cells, nucleic acid,cationic polymer and the like, the molar ratio (negative charge:positivecharge ratio) of each charged group of a nucleic acid, oligonucleic acidor derivative thereof and a cationic polymer, or cationic lipid oraggregate containing the same used in the freeze-dried product of thepresent invention may be 1:0.8 to 1:100, preferably 1:1 to 1:50 and morepreferably 1:1.2 to 1:30. The blending ratio between a nucleic acid,oligonucleic acid or derivative thereof and a cationic polymer, orcationic lipid or aggregate containing the same refers to the molarratio of each charged group, and more specifically indicates the molarratio of negative charge attributable to phosphate anions of a nucleicacid, oligonucleic acid or derivative thereof to positive charge of acationic polymer, or cationic lipid or aggregate containing the same, orfunctional groups able to be positively charged.

Although varying according to the types of target cells, nucleic acid,anionic polymer and the like, the molar ratio (negative charge:negativecharge ratio) of each charged group between a nucleic acid, oligonucleicacid or derivative thereof to anionic polymer used in the freeze-driedproduct of the present invention may be 1:0.2 to 1:1000, preferably1:0.2 to 1:100 and more preferably 1:1 to 1:60. The blending ratiobetween nucleic acid, oligonucleic acid or derivative thereof andanionic polymer refers to the molar ratio of each charged group, andmore specifically indicates the molar ratio of negative chargeattributable to phosphate anions of a nucleic acid, oligonucleic acid orderivative thereof to negative charge of the anionic polymer orfunctional groups able to be negatively charged.

For example, in the case of using hyaluronic acid for the anionicpolymer, the blending ratio of nucleic acid to hyaluronic acid is 1:0.2to 1:1000, preferably 1:0.2 to 1:100 and more preferably 1:1 to 1:60.

For example, in the case of using a PEG derivative having carboxyl sidechains for the anionic polymer, the blending ratio of nucleic acid toPEG derivative having carboxyl side chains may be 1:0.2 to 1:1000,preferably 1:0.2 to 1:100 and more preferably 1:1 to 1:60.

In particular, in the case of using polyethyleneimine for the cationicpolymer and hyaluronic acid for the anionic polymer, the blending ratioof nucleic acid to polyethyleneimine to hyaluronic acid is 1:2 to 60:1to 240, preferably 1:4 to 24:1 to 160, more preferably 1:7 to 20:2 to 60and particularly preferably 1:8 to 14:2 to 32.

In particular, in the case of using polyethyleneimine for the cationicpolymer and a PEG derivative having carboxyl side chains for the anionicpolymer, the blending ratio of nucleic acid to polyethyleneimine to PEGderivative having carboxyl side chains is 1:2 to 60:1 to 240, preferably1:4 to 24:2 to 160, more preferably 1:7 to 20:2 to 60 and particularlypreferably 1:8 to 14:4 to 32.

In particular, in the case of using lipofectamine (liposome containing3:1 w/w mixture of DOSPA and DOPE) as an aggregate containing cationiclipid and hyaluronic acid for the anionic polymer, the blending ratio ofnucleic acid to lipofectamine to hyaluronic acid is 1:1 to 50:0.2 to240, preferably 1:1.2 to 48:0.2 to 160, more preferably 1:1.5 to 30:0.5to 60, and particularly preferably 1:1.8 to 16:1 to 32.

In particular, in the case of using lipofectamine as an aggregatecontaining cationic lipid and a PEG derivative having carboxyl sidechains for the anionic polymer, the blending ratio of nucleic acid tolipofectamine to PEG derivative having carboxyl side chains is 1:1 to50:0.1 to 160, preferably 1:1.2 to 48:0.2 to 160, more preferably 1:1.5to 30:0.5 to 60 and particularly preferably 1:1.8 to 16:2 to 32.

Although preferable blending ratios of nucleic acid, oligonucleic acidor a derivative thereof; cationic polymer or cationic lipid or aggregatecontaining the same; and anionic polymer contained in the freeze-driedproduct of the present invention are as described above, since optimumconditions fluctuate according to the number and type of cellsintroduced with nucleic acid and the like, the blending ratio can besuitably determined by a person with ordinary skill in the art accordingto the types of cells and nucleic acid used.

The freeze-dried product of the present invention can be prepared by astep of forming a complex by mixing the aforementioned nucleic acid,oligonucleic acid or derivative thereof, cationic polymer or cationiclipid or aggregate containing the same, and anionic polymer in theblending ratios described above, and a step of freeze-drying thecomplex. The order of mixing is preferably [1] nucleic acid,oligonucleic acid or derivative thereof, [2] cationic polymer orcationic lipid or aggregate containing the same, and [3] anionicpolymer; or [1] nucleic acid, oligonucleic acid or derivative thereof,[2] anionic polymer, and [3] cationic polymer or cationic lipid oraggregate containing the same. The complex is formed as a result of thenucleic acid, oligonucleic acid or derivative thereof bonding with thecationic polymer or cationic lipid or aggregate containing the same byionic bonding, followed by the cationic polymer or cationic lipid oraggregate containing the same bonding with the anionic polymer by ionicbonding. Alternatively, depending on the blending composition of eachcomponent, the outer shell of such a complex may be coated mainly withthe anionic polymer resulting in the formation of a mode having anegative surface potential.

Next, the resulting complex is freeze-dried. Freeze-drying can becarried out under ordinary freeze-drying conditions such as underconditions consisting of drying under reduced pressure (preferably 5 to100 mmHg and more preferably 10 mmHg) at an external temperature of −78to 60° C. and preferably −30 to 40° C. The time required for dryingvaries according to the degree of depressurization and the amount ofsolvent, and is normally completed in 1 hour to 2 days.

The freeze-dried product of the present invention prepared in thismanner can be used for various types of gene therapy, antisense therapyor introduction of a specific gene into humans and animals, and for theproduction of controlled, knockdown and knockout experimental animalsand cells. More specifically, the freeze-dried product of the presentinvention can be used after converting to a rehydrate by suspending ordissolving in a solvent such as water, physiological saline, buffer,glucose solution or liquid medium prior to use. When rehydrating, thefreeze-dried product is suspended or diluted using 100 to 10000 times(weight ratio) more of solvent than the nucleic acid, oligonucleic acidor derivative thereof, for example. Since a different amount ordifferent type of solvent can be used from that prior to freeze-drying,comparatively high concentrations of suspensions or solutions (such asliquids containing 1 mg of DNA in 1 ml of liquid), which were difficultto prepare in the past, can be prepared easily.

Thus rehydrated freeze-dried product of the present invention can beused for introducing a nucleic acid and the like into cells by using anyarbitrary method normally used to introduce a nucleic acid, oligonucleicacid or derivatives thereof into cells of the living body. Specific oneincludes an ex vivo method in which target cells placed in wells afterhaving been removed from the body are treated with the rehydratedfreeze-dried product of the present invention to introduce a gene orantisense nucleic acid and the cells are returned to the body to expressthe intended gene; or an in vivo and in situ method for directlyintroducing a gene or antisense nucleic acid, and so forth.

In addition, the freeze-dried product of the present invention can alsobe administered without rehydrating by means such as contacting withcells into which a nucleic acid and the like is to be introduced,subcutaneously transplanting into an animal in which a nucleic acid isto be introduced, or transplanting into, onto the surface of, or in thevicinity of a target tissue in which a nucleic acid is to be introduced.

Although varying according to the type of introduction method asdescribed above or the disease, the amount of the freeze-dried productof the present invention applied to cells in terms of the amount of, forexample, nucleic acid, oligonucleic acid or derivative thereof in an exvivo method or in situ method is 0.2 to 10 μg/10⁴ to 10⁷ cells per 1 to2 cm diameter well, and in the case of an in vivo method, 5 to 1000μg/cm³ of tumor, for example, in the case of local administration into atumor, although varying considerably according to the administrationsite, and for example, 0.1 μg to 100 mg/organ in the case ofadministration into an organ such as the urinary bladder or 0.1 ng to 10mg/kg of body weight in the case of systemic administration.

Any method employed in the field of gene therapy can be used as an invivo method for directly administering into the body, examples of whichinclude injecting a rehydrated freeze-dried product of the presentinvention intravenously, subcutaneously, intramuscularly,intraperitoneally, into a tumor or the vicinity of a tumor, inhalingthrough the nasal cavity, oral cavity or lungs, directly injecting intothe urinary bladder or rectum, directly administering into tissue at thesite of a lesion or a nearby blood vessel or implanting by supporting ona porous body or non-woven fabric and the like such as a gelatinoussubstance or sponge.

In addition, even when using a hydrate of the freeze-dried product ofthe present invention without rehydrating, the freeze-dried product inan amount as previously described can be used by an ex vivo method, insitu method or in vivo method as described above.

In the freeze-dried product of the present invention, together with theanionic polymer being neutralized, the neutralizing action of thepositive charge of a complex of an ordinary nucleic acid, oligonucleicacid or derivative thereof and a cationic polymer or cationic lipid oraggregate containing the same is retained even after being administeredinto the living body or cells. As a result, interactions such asagglutination occurring between the complex and serum proteins, bloodcells or the extracellular matrix and the like are inhibited. Inaddition, since enzymatic degradation of nucleic acids, oligonucleicacids or derivatives thereof is inhibited, nucleic acids are efficientlytaken up by cells and expressed with high efficiency.

As has been described above, a hydrate of the freeze-dried product ofthe present invention can be used as a preparation or reagent forintroducing a nucleic acid, oligonucleic acid or derivative thereof, oras a kit for introducing a nucleic acid, oligonucleic acid or derivativethereof.

The following provides a more detailed explanation of the presentinvention through Examples thereof. Furthermore, these Examples areprovided for the purpose of explaining the present invention, and do notlimit the invention in any way.

EXAMPLE 1 Gene Expression by Freeze-Dried Product of aPlasmid/Polyethyleneimine (PEI)/Hyaluronic Acid (HA) Complex

A freeze-dried complex comprised of the three components of a gene, PEIand HA was incubated with mouse melanoma cell line derived B16 toconfirm the expression of luciferase gene.

The same plasmid as that described in Non-Patent Document 6(Biomacromolecules, Vol. 7, pp. 1274-1279) was used for the luciferaseplasmid. Linear PEI (Polyscience, Inc.) having a molecular weight ofMw=25,000 was used for the PEI. Microbial hyaluronic acid(Nacalai-Tesque) was used for the HA. Phosphate Buffered Salts (tablet,Roman Industries) dissolved in ion exchange distilled water was used asPBS. This applies similarly to the following examples as well.

[Operation Procedure]

[1] B16 cells were seeded into a 24-well multiplate two days prior togene introduction and then incubated for two nights using EMEM medium.[2] 2 μl of an aqueous solution containing 1.3 μg of luciferase plasmidwas mixed with 2 μl of an aqueous solution of PEI to a +/−ratio (chargemolar ratio) of 8 on the day prior to gene introduction, and afterpipetting several times, 4 μl of HA solutions of various concentrationswere added and stirred well followed by freezing at −30° C.Subsequently, freeze-drying was carried out to prepare freeze-driedproducts of the present invention. In addition, a freeze-dried productwas prepared using the same method with the exception of changing themixing order of HA and PEI.[3] After removing the cultured medium, 500 μl of EMEM containing, 10%FBS, 25 U of penicillin and 25 μg of streptomycin was placed in thewells.[4] 16 μl of PBS was mixed with the freeze-dried products prepared in[2] followed by incubating for 1 hour and adding to the wells.[5] The mixtures were incubated for 4 hours at 37° C. in 5% CO₂ and 95%air.[6] The medium was replaced with fresh EMEM containing 10% FBS, 25 U ofpenicillin and 25 μg of streptomycin followed by incubating for 20 hoursat 37° C.[7] After incubating for 20 hours, the medium was removed followed bythe addition of 200 μl of PicaGene cell lysis solution to each well.After allowing to stand for about 20 minutes, the cells were separatedfrom the wells and recovered in microtubes.[8] Following centrifugation (15,000 rpm, 1 minute), the supernatant wasassayed for luciferase. The luciferase assay was carried out accordingto the procedure provided with the PicaGene Luminescence Kit.

Furthermore, the cell lysis solution was used directly for proteinassay. The protein assay was carried out using a protein assay kit(Bio-Rad).

For the sake of comparison, gene expression was investigated forfreeze-dried products and non-freeze-dried products to which HA was notadded.

[Results]

The results are shown in the graph below. In the graph, values inparentheses indicate the ratio of PEI cations and HA anions to plasmidanions, and more specifically, the molar ratio of the charges of PEI andHA to DNA.

In the case of the freeze-dried plasmid/PEI binary complex, expressionwas 1/1000 or less that prior to freeze-drying, and in contrast tohardly any expression being observed, in the case of addition of HA,high expression was observed, and in the case of freeze-drying aftermixing plasmid, PEI and HA at a ratio of 1:8:16 (in terms of charge),expression efficiency with an additional 11% or more higher than theplasmid/PEI binary complex prior to freeze-drying was demonstrated.

TABLE 1

EXAMPLE 2 Effects of Mixing Order

Freeze-dried products were obtained by mixing with addition of PEI afterfirst adding HA to luciferase plasmid in [2] of Example 1 followed byevaluating in the same manner as Example 1.

[Results]

The results are shown in the graph below. In the graph, values inparentheses indicate the ratio of PEI cations and HA anions to plasmidanions and more specifically, indicate the molar ratio of PEI and HA toDNA.

In contrast to hardly any gene expression being observed in the case ofthe freeze-dried plasmid/PEI binary complex, high expression wasobserved in the case of addition of HA, and in the case of freeze-dryingafter mixing plasmid, PEI and HA at a ratio of 1:8:16 (in terms ofcharge), expression efficiency with an additional 26% or more higherthan the plasmid/PEI binary complex prior to freeze-drying wasdemonstrated. On the basis of these results, high expression was clearlyobserved even if the order of mixing is changed.

TABLE 2

EXAMPLE 3 Gene Expression by Freeze-Dried Product ContainingPlasmid/PEI/PEG Derivative Having Carboxyl Side Chains (PEG-C)

In Example 3, PEG-C having a molecular weight of about 10,000 andcontaining about 18 carboxyl groups per molecule was used as anionicpolymer after synthesizing according to the method described inNon-Patent Document 1 (J. Biomater. Sci. Polymer Edn., Vol. 14, pp.515-531 (2003)).

A freeze-dried three-component complex comprised of a gene, PEI andPEG-C was incubated with mouse melanoma cell line B16 followed toconfirm the expression of luciferase gene.

[Procedure]

[1] B16 cells were seeded into a 24-well multiplate two days prior togene introduction and then incubated for two nights using EMEM medium.[2] 12.5 μl of an aqueous solution containing 1.3 μg of luciferaseplasmid was mixed with 12.5 μl of an aqueous solution of PEI to a+/−ratio (charge molar ratio) of 8 on the day prior to geneintroduction, and after pipetting several times, 25 μl of PEG-Csolutions of various concentrations were added and stirred well followedby freezing at −30° C. Subsequently, freeze-drying was carried out toprepare freeze-dried products of the present invention.[3] After removing the cultured medium, 500 μl of EMEM containing, 10%FBS, 25 U of penicillin and 25 μg of streptomycin was placed in thewells.[4] 50 μl of PBS was mixed with the freeze-dried products prepared in[2] followed by incubating for 1 hour and adding to the wells.[5] The mixtures were incubated for 4 hours at 37° C. in 5% CO₂ and 95%air.[6] The medium was replaced with fresh EMEM containing 10% FBS, 25 U ofpenicillin and 25 μg of streptomycin followed by incubating for 20 hoursat 37° C.[7] After incubating for 20 hours, the medium was removed followed bywashing the cells once with PBS and adding 200 μl of PicaGene cell lysissolution to each well. After allowing to stand for about 20 minutes, thecells were separated from the wells and recovered in microtubes.[8] Following centrifugation (15,000 rpm, 1 minute), the supernatant wasassayed for luciferase. The luciferase assay was carried out accordingto the procedure provided with the PicaGene Luminescence Kit.

Furthermore, the cell lysis solution was used directly for proteinassay. The protein assay was carried out using a protein assay kit(Bio-Rad).

In addition, for the sake of comparison, an experiment was carried outin the same manner for a freeze-dried product obtained by adding aneutral polymer PEG, having about the same molecular weight as PEG-C butnot having a charge, in an amount equal to that of PEG-C.

[Results]

The results are shown in the graph below. In the graph, values inparentheses indicate the ratio of PEI cations and PEG-C anions toplasmid anions, and specifically, the molar ratio of the charges of PEIand PEG-C to DNA.

There was hardly any expression observed in the case of the freeze-driedplasmid/PEI binary complex. In addition, there was hardly any expressionobserved even following addition of the uncharged PEG. On the otherhand, in the case of the freeze-dried product of the present inventionto which was added PEG-C, high expression was observed that was nearly60% of the non-freeze-dried original plasmid/PEI binary complex.

TABLE 3

EXAMPLE 4 Gene Expression by Plasmid/Lipofectamine/HA Freeze-DriedProduct

In this example, lipofectamine manufactured by Invitrogen was used forthe lipofectamine.

A freeze-dried three-component complex comprised of a gene,lipofectamine and HA was incubated with mouse melanoma cell line derivedB16 to confirm the expression of luciferase gene.

[Operation Procedure]

[1] B16 cells were seeded into a 24-well multiplate two days prior togene introduction and then incubated for two nights using EMEM medium.[2] 12.5 μl of an aqueous solution containing 1.3 μg of luciferaseplasmid was mixed with 12.5 μl of an aqueous solution of lipofectamineto a weight ratio of luciferase plasmid to lipofectamine of 8 on the dayprior to gene introduction, and after pipetting several times, 25 μl ofHA solutions of various concentrations were added and incubated for 30minutes followed by freezing at −30° C. Subsequently, freeze-drying wascarried out to prepare freeze-dried products of the present invention.[3] After removing the cultured medium, 500 μl of EMEM containing 25 Uof penicillin and 25 μg of streptomycin was placed in the wells.[4] 50 μl of PBS was mixed with the freeze-dried products prepared in[2] followed by incubating for 45 minutes and adding to the wells.[5] The mixtures were incubated for 4 hours at 37° C. in 5% CO₂ and 95%air.[6] 100 μl of fresh EMEM containing 25 U of penicillin and 25 μg ofstreptomycin and 400 μl of FBS were added followed by incubating for 20hours at 37° C.[7] After incubating for 24 hours, the medium was removed followed bywashing the cells once with PBS and adding 200 μl of PicaGene cell lysissolution to each well. After allowing to stand for about 20 minutes, thecells were separated from the wells and recovered in microtubes.[8] Following centrifugation (15,000 rpm, 1 minute), the supernatant wasassayed for luciferase. The luciferase assay was carried out accordingto the procedure provided with the PicaGene Luminescence Kit.

Furthermore, the cell lysis solution was used directly for proteinassay. The protein assay was carried out using a protein assay kit(Bio-Rad).

For the sake of comparison, gene expression was investigated forfreeze-dried and non-freeze-dried products to which HA was not added.

In addition, a similar study was carried out by carrying out theincubation of cells and DNA complex of [3] in EMEM medium containing 80%FBS. In this case, instead of adding FBS in [6], 1500 μl of EMEM onlycontaining 25 U of penicillin and 25 μg of streptomycin was added.

[Results]

The results are shown in the graph below. In the graph, values inparentheses indicate the weight ratio of lipofectamine and HA toplasmid.

In the case of gene introduction in medium not containing FBS,expression in the case of the freeze-dried plasmid/lipofectamine binarycomplex was 1/3000 or less that prior to freeze-drying, and hardly anyexpression was observed. In contrast, in the case of addition of HA,expression of about 55% of the non-freeze-dried originalplasmid/lipofectamine binary complex was demonstrated.

In the presence of 80% serum, although expression by the freeze-driedplasmid/lipofectamine binary complex decreased further, the freeze-driedplasmid/lipofectamine/HA tertiary complex exhibited high expression ofabout 57% of the non-freeze-dried original plasmid/lipofectamine binarycomplex.

TABLE 4

EXAMPLE 5 Gene Expression by Plasmid/Lipofectamine/PEG-C Freeze-DriedProduct

The same experiment as Example 4 was carried out using PEG-C having acharge ratio of 16 relative to the plasmid DNA instead of HA. Morespecifically, instead of adding 25 μl of HA solution as in Example 4, anamount of PEG-C was dissolved in water so that the charge ratio was 16times that of the plasmid DNA as described in the following graph andadded at a final volume of 25 μl.

Gene introduction was carried out in serum-free medium or mediumcontaining 80% serum.

[Results]

The results are shown in the graph below. In the graph, values inparentheses indicate the weight ratio of lipofectamine and PEG-C to DNA.

In contrast to hardly any expression being observed in the case offreeze-dried DNA/lipofectamine binary complex, high expression wasdemonstrated following addition of PEG-C, and in the presence of 80%serum, the plasmid/lipofectamine/PEG-C tertiary complex demonstratedhigh expression efficiency four times or more the non-freeze-driedoriginal plasmid/lipofectamine binary complex.

TABLE 5

EXAMPLE 6 Effect of Changes in Concentration Before and afterFreeze-Drying

A liquid was prepared at a low concentration using 10 times the amountof solvent as in [2] of Example 5 followed by mixing, freeze-drying,adding 50 μl of PBS in the same manner as [4] of Example 5, andsimilarly evaluating the rehydrated product.

[Results]

The results are shown in the graph below. In the graph, values inparentheses indicate the weight ratio of lipofectamine and PEG-C to DNA.

In contrast to hardly any expression being observed in the case offreeze-dried DNA/lipofectamine binary complex, with expression of 1/1000or less that prior to freeze-drying, high expression was observed in thecase PEG-C was added, even when concentrated after freeze-drying. In thepresence of 80% serum, plasmid/lipofectamine/PEG-C tertiary complexdemonstrated high expression efficiency nearly 30% more than thenon-freeze-dried original plasmid/lipofectamine binary complex.

On the basis of these results, arbitrary concentrations of complexsuspensions or solutions capable of facilitating gene introduction wereconfirmed to be able to be prepared as a result of freeze-drying.

TABLE 6

EXAMPLE 7 Administration of Freeze-Dried Solid DNA Complex to LivingOrganism [Procedure]

50 μl of a TE buffer solution of luciferase-encoded plasmid (0.8 mg/ml)was diluted with 400 μl of water followed by the addition of 100 μl ofaqueous hyaluronic acid solution (5.8 mg/ml) and finally the addition of50 μl of PEI solution (1.25 mg/ml). Thirty minutes after mixing thethree components, the mixture was freeze-dried at −30° C. followed byfreeze-drying to obtain a solid complex.

4.72×10⁶ mouse melanoma cell line derived B16 suspended in 100 μl ofmedium*1 was subcutaneously transplanted into 5-week-old, male ddY mice.When the tumors had reached 6 to 8 mm, an incision was made in the tumorportion under anesthesia and the solid DNA/PEI/HA complex describedabove was implanted within the tumor followed by suturing the incision.

Two days later, the mice were sacrificed with ether, the tumors and skinwere excised and then homogenized in 1 ml of cell lysis solution*2.Subsequently, centrifugation was carried out for 20 minutes at 10,000rpm and 4° C., and a substrate (Promega, 20 μl) was added to thesupernatant (5 μl) to measure the luminescence of the luciferase for 30seconds with a luminometer.

Total protein was quantified by adding 20 μl of supernatant of eachsample diluted 1/80 to 1 ml of protein quantification reagent (Bio-Rad)followed by measuring absorbance at a wavelength of 595 nm 20 minuteslater.

[Results]

The solid DNA complex demonstrated extremely high expression within thetumor (results are shown in the table below).

*1 medium; EMEM medium (containing 10% FBS, penicillin G sodium (100units/ml) and streptomycin sulfate (0.1 mg/ml))*2 cell lysis solution; 0.05% Triton X-100, 2 mM EDTA and 0.1 M Tris-HCl(pH 7.5)

TABLE 7

EXAMPLE 8 Gene Introduction into Cells on a Culture Plate UsingFreeze-Dried DNA Complex [Procedure]

1.56 μl of a solution of luciferase-encoded plasmid (0.8 mg/ml) wasdiluted with 12.5 μl of water followed by the addition of 1.56 μl of PEIsolution (1.25 mg/ml) and finally the addition of 3.56 μl of aqueoushyaluronic acid solution (5.8 mg/ml). After mixing the three components,the mixture was placed in the wells of a culture plate, frozen at −30°C. 30 minutes later, and subsequently freeze-dried. A mixture to whichhyaluronic acid was not added was similarly freeze-dried. In addition, amixture using a fresh suspension without freeze-drying wassimultaneously compared.

1.2×10⁵ mouse melanoma cell line derived B16 suspended in 300 μl ofmedium*1 was seeded into the wells containing freeze-dried DNA complex.1 ml of medium was added 4 hours later and was replaced with 1 ml offresh medium 20 hours later. 24 hours later, 200 μl of cell lysissolution (Promega) were added and the cells were harvested followed bycentrifuging for 1 minute at 15,000 rpm and 4° C., adding a substrate(Promega, 20 μl) to the supernatant (5 μl), and measuring theluminescence of the luciferase for 30 seconds with a luminometer.

Total protein was quantified by adding 20 μl of supernatant of eachsample diluted 1/5 to 1 ml of protein quantification reagent (Bio-Rad)followed by measuring absorbance at a wavelength of 595 nm 20 minuteslater.

*1 medium; EMEM medium (containing 10% FBS, penicillin G sodium (100units/ml) and streptomycin sulfate (0.1 mg/ml)).

[Results]

Although nearly all gene introduction activity had disappeared followingfreeze-drying in the absence of addition of hyaluronic acid (HA), in thecase of addition of HA, high activity similar to that of the freshsuspension was demonstrated even after freeze-drying (results are shownin the table below).

TABLE 8

EXAMPLE 9 In Vivo Gene Introduction Using Suspension of DNA ComplexConcentrated by Freeze-Drying [Operation Procedure]

62.5 μl of a solution of luciferase-encoded plasmid (0.8 mg/ml) wasdiluted with 0, 500, 2000 or 8000 μl of water followed by the additionof 125 μl of aqueous hyaluronic acid solution (5.8 mg/ml) and finallythe addition of 62.5 μl of PEI solution (1.25 mg/ml). Thirty minutesafter mixing the three components, the mixtures were frozen at −30° C.followed by freeze-drying.

The freeze-dried DNA complexes were resolvated with 250 μl of 5%glucose.

4.72×10⁶ mouse melanoma cell line derived B16 suspended in 100 μl ofmedium*1 was subcutaneously transplanted into 5-week-old, male ddY mice.When the tumors had reached 6 to 8 mm, a suspension of resolvated DNAcomplex was administered into a tail vein of the mice.

The mice were exsanguinated under ether anesthesia 24 hours laterfollowed by excision of the tumor, liver and lungs and homogenizing in 1ml of cell lysis solution*2. Subsequently, centrifugation was carriedout for 20 minutes at 10,000 rpm and 4° C., and a substrate (Promega, 20μl) was added to the supernatant (5 μl) to measure the luminescence ofthe luciferase for 30 seconds with a luminometer.

Total protein was quantified by adding 20 μl of supernatant of eachsample diluted 1/80 to 1 ml of protein quantification reagent (Bio-Rad)followed by measuring absorbance at a wavelength of 595 nm 20 minuteslater.

*1 medium; EMEM medium (containing 10% FBS, penicillin G sodium (100units/ml) and streptomycin sulfate (0.1 mg/ml))*2 cell lysis solution; (0.05% Triton X-100, 2 mM EDTA and 0.1 MTris-HCl (pH 7.5))

[Results]

The results are shown in the graph below. Concentrations in the graphindicate the final concentrations of DNA at the time of complexpreparation as the nucleic acid base concentrations. In the case offreeze-drying following addition of hyaluronic acid (HA), higher geneexpression was observed the lower the concentration of DNA at the timeof preparation, and remarkably high luciferase activity was demonstratedwithin the tumor in particular.

TABLE 9

EXAMPLE 10 Gene Expression Inhibitory Effects by siRNA ComplexFreeze-Dried on a Culture Plate [Operation Procedure]

25 μl of a protamine aqueous solution (78 μg/ml) was added to 25 μl ofan aqueous solution of anti-luciferase siRNA (Invitrogen, 21.28 μg/ml)followed by the addition of 50 μl of hyaluronic acid solution (53.7μg/ml or 107.5 μg/ml). After mixing the three components, the mixtureswere placed in the wells of a culture plate, frozen at −30° C. for 30minutes and subsequently freeze-dried.

1.2×10⁵ mouse melanoma cell line derived B16 suspended in 100 μl ofmedium*1 was seeded onto a culture plate followed by the addition of 1ml of medium 4 hours later and the addition of a mixture of 25 μl ofpDNA solution (50 μg/ml) and 25 μl of PEI solution (78 μg/ml). Moreover,the medium was replaced with 1 ml of fresh medium 20 hours later.

24 hours later, 200 μl of cell lysis solution (Promega) was added andthe cells were harvested followed by centrifuging for 1 minute at 15,000rpm and 4° C., adding substrate (Promega, 20 μl) to the supernatant (5μl), and measuring the luminescence of the luciferase for 30 secondswith a luminometer.

Total protein was quantified by adding 20 μl of supernatant of eachsample diluted 1/5 to 1 ml of protein quantification reagent (Bio-Rad)followed by measuring absorbance at a wavelength of 595 nm 20 minuteslater.

*1 medium; EMEM medium (containing 10% FBS, penicillin G sodium (100units/ml) and streptomycin sulfate (0.1 mg/ml)).

Mixtures to which protamine and hyaluronic acid were not added were alsosimilarly freeze-dried.

[Results]

Expression of luciferase was significantly inhibited in cells culturedon plate which had been freeze-dried following the addition of protamine(PRT) and hyaluronic acid (HA) (refer to graph below).

TABLE 10

EXAMPLE 11 Size of DNA Complexes Prepared at Different ConcentrationsFollowing Rehydration [Operation Procedure]

1.5 μl of the same solution of luciferase plasmid as used in Example 1(0.8 mg/ml) was diluted with 0, 12.5 or 200 μl of water followed by theaddition of 3 μl of aqueous hyaluronic acid solution (5.8 mg/ml) andfinally the addition of 1.5 μl of PEI solution (1.25 mg/ml). Thirtyminutes after mixing the three components, the mixtures were frozen at−30° C. followed by freeze-drying.

The freeze-dried DNA complexes were rehydrated with 6 μl of waterfollowed by the addition of 800 μl of water 30 minutes later andmeasuring the size of the complexes with a zeta analyzer (MalvernInstruments).

[Results]

The percentages of formed complex particles measuring 0 to 100 nm andthe percentages of formed complex particles measuring 100 to 200 nm areshown in the graph below. The values shown after the names of thecomponents indicate the final DNA concentration at the time of complexpreparation as nucleic acid base concentrations.

There were hardly any fine particles observed after rehydration in thecase of not adding hyaluronic acid (HA). On the other hand, in the caseof freeze-drying following the addition of hyaluronic acid, numerousparticles were observed to maintain their small size even afterrehydration. In addition, although DNA concentrations followingrehydration were the same in all cases, the percentage of fine particleswas observed to be greater in the case where the complexes were preparedunder the more dilute conditions.

TABLE 11

1. A freeze-dried product of a complex containing a nucleic acid,oligonucleic acid or derivative thereof; a cationic polymer, or cationiclipid or aggregate containing the same; and, an anionic polymer.
 2. Thefreeze-dried product according to claim 1, wherein the molar ratio ofeach charged group (ratio of negative charge to positive charge) of acomplex containing a nucleic acid, oligonucleic acid or derivativethereof and the cationic polymer, or cationic lipid or aggregatecontaining the same is 1:0.8 to 1:100.
 3. The freeze-dried productaccording to claim 1, wherein the molar ratio of each charged group(ratio of negative charge to negative charge) of a nucleic acid,oligonucleic acid or derivative thereof and an anionic polymer is 1:0.2to 1:1000.
 4. The freeze-dried product according to claim 1, wherein thecationic polymer is a positively charged, naturally-occurring orsynthetic polymer having a molecular weight of about 1,000 to 3,000,000and having a plurality of functional groups capable of forming a complexwith DNA in water in a molecule.
 5. The freeze-dried product accordingto claim 4, wherein the cationic polymer is selected from positivelycharged proteins and polypeptides; positively charged dendrimers;positively charged synthetic polymers; and positively chargedpolysaccharide derivatives or salts and combinations thereof.
 6. Thefreeze-dried product according to claim 1, wherein the cationic lipid isDC-Chol (3β-(N—(N′,N′-dimethylaminoethane) carbamoyl) cholesterol), DDAB(N,N-distearyl-N,N-dimethylammonium bromide), DMRI(N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide), DODAC (N,N-dioleyl-N,N-dimethylammonium chloride), DOGS(diheptadecylamidoglycylspermidine), DOSPA(N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate), DOTAP(N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride) or DOTMA(N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride).
 7. Thefreeze-dried product according to claim 1, wherein the anionic polymeris a negatively charged, naturally-occurring or synthetic polymercontaining an anionic group in a molecule thereof, having a molecularweight of about 500 to 4,000,000, and having a plurality of functionalgroups in a molecule thereof capable of forming a complex with apolycation in water.
 8. The freeze-dried product according to claim 7,wherein the anionic polymer is selected from polysaccharides andderivatives thereof having functional groups selected from a carboxylgroup, —OSO₃H group, —SO₃H group, phosphate group and salts thereof;polyamino acids containing an amino acid residue having negativelycharged side chains; PEG derivatives having carboxyl side chains;synthetic polymers having functional groups selected from a carboxylgroup, —OSO₃H group, —SO₃H group, phosphate group and salts thereof;polymers having functional groups selected from a carboxyl group, —OSO₃Hgroup, —SO₃H group, phosphate group and salts thereof, as well asoptionally substituted amino groups, ammonium groups or salts thereof,and combinations thereof.
 9. The freeze-dried product according to claim1, wherein the aggregate containing a cationic lipid is an aggregatecontaining DOSPA(N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoroacetate) and the anionic polymer is hyaluronic acid.
 10. Thefreeze-dried product according to claim 1, wherein the aggregatecontaining a cationic lipid is an aggregate containing DOSPA(N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoroacetate), and the anionic polymer is a PEG derivative havingcarboxyl side chains.
 11. The freeze-dried product according to claim 1,wherein the cationic polymer is polyethyleneimine; and the anionicpolymer is hyaluronic acid.
 12. The freeze-dried product according toclaim 1, wherein the cationic polymer is polyethyleneimine; and theanionic polymer is a PEG derivative having carboxyl side chains.
 13. Thefreeze-dried product according to claim 11, wherein the blending ratioof nucleic acid, oligonucleic acid or derivative thereof topolyethyleneimine to hyaluronic acid is 1:4 to 24:1 to 160 (molar ratioof each charged group).
 14. The freeze-dried product according to claim12, wherein the blending ratio of nucleic acid, oligonucleic acid orderivative thereof to polyethyleneimine to PEG derivative havingcarboxyl side chains is 1:4 to 24:2 to 160 (molar ratio of each chargedgroup).
 15. The freeze-dried product according to claim 9, wherein theblending ratio of nucleic acid, oligonucleic acid or derivative thereofto aggregate containing DOSPA to hyaluronic acid is 1:1.2 to 48:0.2 to160 (molar ratio of each charged group).
 16. The freeze-dried productaccording to claim 10, wherein the blending ratio of nucleic acid,oligonucleic acid or derivative thereof to aggregate containing DOSPA toPEG derivative having carboxyl side chains is 1:1.2 to 48:0.2 to 160(molar ratio of each charged group).
 17. A method for preparing thefreeze-dried product according to claim 1, comprising a step of forminga complex by mixing a nucleic acid, oligonucleic acid or derivativethereof; a cationic polymer, or cationic lipid or aggregate containingthe same; and an anionic polymer, and a step of freeze-drying thecomplex.
 18. A preparation or reagent containing the freeze-driedproduct according to claim 1 for introducing a nucleic acid,oligonucleic acid or derivative thereof.
 19. A kit containing thefreeze-dried product according to claim 1 for introducing a nucleicacid, oligonucleic acid or derivative thereof.
 20. A method forintroducing a nucleic acid, oligonucleic acid or derivative thereof intocells that uses the freeze-dried product according to claim
 1. 21. Themethod according to claim 20, comprising a step of rehydrating thefreeze-dried product in a solvent before introducing a nucleic acid,oligonucleic acid or derivative thereof.
 22. The method according toclaim 20, wherein the freeze-dried product is used without rehydrating.